CZ248295A3 - In vitro stimulating method of non-keratinocytic epithelial cells and growth factor of keratinocytes for treating diseases - Google Patents

Abstract

Based on extensive in vivo studies in animals, it has now been discovered that KGF stimulates proliferation, growth and differentiation in various cells of epithelial tissue, besides keratinocytes. This better understanding of the biological effects of KGF in vivo enables the use of this polypeptide as a therapeutic agent, suitably formulated in a pharmaceutical composition, for the specific treatment of disease states and medical conditions afflicting tissues and organs such as the dermal adnexae, the liver, the lung, and the gastrointestinal tract.

Description

Technical field

The present invention relates to the use of keratinocyte growth factor (KGF) for stimulating proliferation of growth and differentiation of cells other than keratinocytes. Further, the invention relates to the use of KGF for the treatment of diseases in patients in whom it is beneficial to achieve a biological effect of regenerating damaged and diseased cells and tissues.

Background Art

Progress in biotechnology in recent years has led to the development and therapeutic use of new recombinant polypeptides, such as erythropoietin and granulocyte colony stimulating factor, for the benefit of many human patients. A number of other polypeptides have been discovered since then, exhibiting biological activity in vitro. However, the identification of the biological effects of these factors and the characterization of the target cells targeted by these factors is important to identify potential therapeutic applications in humans.

KGF is a known mitogen that has recently been found to have specificity for epithelial cells, particularly keratinocytes [Rubin et al., Proc. Nati. Acad. Sci. USA 86: 802-806 (1989); Finch et al., Science, 245: 752-755 (1989) and Marchese et al., J. Cell. Phys. 144: 326-332 (1990)]. KGF mediator RNA expression was found in several stromal fibroblast cell lines derived from epithelial tissues of embryonic, neonatal and adult human origin, and in RNA extracted from normal adult kidney and gastrointestinal tract. Such RNA has not been detected in glial cells, lungs, brain, or various epithelial cell lines, including squamous cell carcinomas, mammary epithelial cells, immortalized bronchial epithelial cells, immortalized keratinocytes, and primary keratinocytes [Finch et al., Science, supra]. . However, KGF is mitogenically effective in the continuous mouse cell line, BALB / MK [Weissman and Aaronson, Cell. 32: 599 (1983) as well as PNAS and Science, supra]. This observation supports the claim that KGF exhibits a paracrine effect in the skin, with KGF being produced in the dermis (but not in the epidermis) and acting on keratinocytes.

Furthermore, through expression of keratin and filagrin genes, KGF has been shown to affect keratinocyte differentiation and maturation [J. Cell. Phys., Supra].

Much of the above-described studies of KGF biological activity have been performed with recombinant KGF, which allowed the study of this factor to be extended. PCT Patent Application WO 90/08771 discloses the purification of KGF from a conditioned human embryonic fibroblast lineage medium, partial sequencing of an isolated polypeptide, gene cloning and expression in bacterial cells (E. coli) to produce recombinant biologically active KGF.

While the role of KGF as a stimulant for keratinocytes in vitro has been clearly identified, many remain unanswered as regards the role of KGF as a growth factor, including the identification of other cell types that KGF may target and the effect of KGF on such cells in vivo. Such information is very important for understanding the overall potential of KGF as a therapeutic agent.

SUMMARY OF THE INVENTION

In short, the invention is based on the discovery of the stimulating effect of KGF on epithelial non-keratinocyte cells. More specifically, KGF has been found to induce in vivo proliferation and differentiation in normal adnexal structures, such as sebocytes and hair follicle cells, in liver cells such as hepatocytes and respiratory mucosal epithelial cells, such as type II pneumocytes. There is also convincing evidence that KGF stimulates the proliferation and growth of intestinal mucosa cells, such as mucin-producing goblet cells in the gastric glands and the small intestine, cryptic cells in the colon, and other epithelial cells in the gut.

These discoveries have significant implications in that they allow KGF to be applied to tissues where there is specific damage or deficiency in cells of these particular types. The following is a description of diseases and conditions that can be treated with KGF in accordance with the teachings of the present invention.

Stimulation of proliferation and differentiation of adnexal structures such as hair follicles, sweat glands and sebaceous glands is critical in the recovery of epidermis and dermis in patients suffering from burns and other partial or full thickness lesions. Surface defects are currently healing by scar formation and restoration of the keratinocyte surface layer, yet complete skin regeneration cannot be achieved. Skin defects involving the entire thickness of the skin, including burns, do not currently repopulate the hair follicles of the sweat glands and sebaceous glands. By using KGF, such repopulation can be achieved.

Epidermolysis bullosa is a defect in the adhesion of the epidermis to the underlying dermis, resulting in frequent open and painful blisters causing serious disease. Accelerated re-epithelization of these lesions, for example using KGF, would result in reduced risk of infection, less pain, and reduced wound care.

Chemotherapy-induced alopecia occurs in patients treated with chemotherapeutic agents to combat malignant tumors. There are currently no drugs available that effectively prevent the killing of hair follicle cells causing transient hair loss.

KGF represents such a composition.

Male-type baldness is the predominant type of baldness and is basically incurable. Progressive hair loss in men and women is a serious cosmetic problem. This condition could be treated using KGF either systemically or topically if the drug could be applied to the scalp, absorbed or spray injected into the scalp using an air gun or other technology.

Although gastric ulcers can be treated with H ₂ antagonists, they are still a major cause of the recurring disease. Their healing consists in the formation of a scar in the rucosal lining. The ability to regenerate glandular mucosa more rapidly, for example by administering KGF, would be a significant therapeutic improvement in the treatment of gastric ulcers.

Although duodenal ulcers, like gastric ulcers, are treatable, the development of therapeutic agents that would result in more complete and faster recovery of the mucosal lining of the duodenum would be a significant advance. It would also be useful for this therapeutic agent to regenerate the ulcer and reduce the frequency of recurrence. KGF has such potential.

Inflammatory bowel diseases such as Crohn's disease (which predominantly affects the small intestine) and ulcerative colitis (which predominantly affects the large intestine) are chronic diseases of unknown aetiology, manifested by the destruction of the mucosal surface, inflammation, scarring and adhesions resulting from healing and considerable morbidity affected patients. Current therapies are based on inflammatory inflammation. Therapeutics such as KGF, stimulating mucosal surface restoration and accelerating healing, could be useful in suppressing the progression of these diseases.

Intestinal toxicity is a major limiting factor for treatment regimens including radiation and chemotherapy. KGF pretreatment could have a cytoprotective effect on the small intestinal mucosa, which would allow to increase doses in such treatments while reducing potential fatal side effects of intestinal toxicity.

KGF treatment has a remarkable effect on mucus production in the gastrointestinal tract. This property may be useful for protecting the intestinal mucosa from damage by ingested harmful substances or in reducing the spread of damage in diseases such as inflammatory bowel disease.

Hyaline membrane disease in preterm neonates results in the absence of type II pneumocyte surfactant formation in the lung, which is manifested by the collapse of the alveoli. Although maturation and secretion by corticosteroid administration can be greatly accelerated in 28-week-old and older fetuses, there is currently no treatment for younger fruits, resulting in significant morbidity and mortality in this population. A therapeutic agent, such as KGF, that induces type II pneumocyte proliferation and differentiation is potentially very useful in the treatment of this disease.

Smoke inhalation is a significant cause of morbidity and mortality in the week following burns, and is caused by the necrosis of the bronchiolar epithelium and the alveolus. A growth factor such as KGF that could stimulate the proliferation and differentiation of these structures and induce their repair and regeneration would be useful in treating such inhalation damage.

Emphysema is the result of progressive loss of alveoli. A growth factor, such as KGF, which could stimulate new growth or which would be cytoprotective to the remaining alveoli would be therapeutically useful. There is currently no effective treatment for this disease.

Cirrhosis of the liver is one of the major causes of morbidity and mortality in addition to viral hepatitis and chronic alcohol consumption. For example, the cytoprotective effect and effect on hepatocyte proliferation and differentiation induced by KGF would increase liver function, resulting in a slowing or disabling of cirrhosis.

Fulminant liver failure is a life-threatening disease that occurs in the last stage of cirrhosis. An agent such as KGF that could induce proliferation of remaining hepatocytes would have a direct beneficial effect on this disease, which is currently treatable only by liver transplantation.

Acute viral hepatitis is often subclinical and has a self-limiting course. However, a minority of patients may experience severe liver damage within a few weeks. A cytoprotective agent such as KGF could be useful in preventing hepatocellular degeneration.

Toxic liver damage induced by acetaminophen, halothane, carbon tetrachloride and other toxins could be improved by growth factor, such as KGF, which exhibits hepatocyte cytoprotective activity.

The subject invention of KGF for therapeutic (or, where appropriate, also prophylactic) treatment of the aforementioned diseases. Another object of the invention is to provide pharmaceutical compositions comprising a therapeutically effective amount of KGF.

BRIEF DESCRIPTION OF THE DRAWINGS FIG

Figure 1 shows the nucleotide sequence of the synthetic oligonucleotide DNA used in the construction of the recombinant KGF expression system used in the in vivo experiments described herein.

Figure 2 shows a partial thickness dermal injury of a rabbit ear that is modified such that the wound penetrates the cartilage to quantify new tissue growth.

Figure 3 shows the reepithelialization of a wound treated with KGF and a control wound using a modified partial thickness dermal injury in the rabbit ear as a model. The total area of regenerating epithelium after KGF treatment is shown on the left, while the percentage of wound reepithelization is shown on the right.

Figure 4 shows the proliferation of basal and suprabasal keratinocytes at the wound margins within 5 days of KGF treatment using rabbit ear injury as a model.

Figure 5 is a histological analysis of bromodeoxyuridine (Brdu) treated hair follicles from wounds treated with KGF and control wounds to show cell proliferation in a modified rabbit model.

Figure 6 shows oil redness of sebaceous glands in KGF and control injuries. injury in the case of the same rabbit model.

Figures 7 and 8 show the micropapillary growth of alveolar septal epithelial cells in the lungs of healthy rats administered KGF intratracheally.

Figures 9 and 10 show a cubic increase in epithelial cells in alveoli of large lung segments following intratracheal administration of KGF to the same healthy rats.

FIG. Figures 11 and 12 show that hyperplastic alveolar epithelial cells forming the lining of alveolar septum of treated rats contain lamellar inclusions of varying size and shape.

FIG. 13A shows hyperplastic effects of KGF treatment in the bronchial epithelium after intratracheal administration of KGF to healthy rats.

FIG. 13B shows the bronchial epithelium of the control group (administered only excipient) of healthy rats corresponding to the test group of FIG. 13A.

Figure 14 shows RNase receptor KGF (KGFR) protection results in adult rat lungs.

FIG. 15 shows dose-dependent increases in gastrointestinal and liver weights following administration of KGF for 4 days.

FIG. 16 shows the dose-dependent increase in protein synthesis in rat liver after 4 days of KGF administration.

FIG. 17 shows increased hepatocyte proliferation in the liver of animals treated with one to seven days of KGF, stannous by labeling Brdu (a) or mitosis (b).

FIG. 18 and 19 report the amount of liver mass obtained after treatment with some hepatectomized KGF rats.

FIG. 20 shows a decrease in serum serum glutamatoxaloacetate tranaminase (SGOT) in carbon tetrachloride poisoned rats after KGF treatment.

FIG. 21 shows the increase in gastric gland length and the amount of mucus produced in the gastric glands of KGF-treated rats for 1-7 days.

FIG. 22 shows the prolongation of duodenal crypts and mucus formation in the small intestine of KGF-treated rats for 1-7 days.

FIG. 23 shows the increase in colonic hyperplastic crypts of KGF-treated rats for 1 to 7 days.

FIG. 24 shows deepening of crypts in colon of rats treated with KGF for 1 to 7 days.

Description of Specific Embodiments of the Invention

In order to identify specific types of epithelial cells in which KGF provides a significant biological effect that is indicative of its potential therapeutic utility, an extensive study in live animals was conducted. KGF was administered to these animals in vivo to monitor and analyze the results. In all of the following examples, recombinant KGF produced as follows was used:

The recombinant KGF gene is isolated from prepuce fibroblast RNA (AG1523) by polymerase reaction (PCR) and the resulting DNA is ligated into the pCFM1156 expression vector between the NdeI and BamHI restriction sites. Plasmid pCFM1156 can be obtained from plasmid pCFM836 by the method described in U.S. Pat.

710,473 (the entire contents of this patent is relevant to the disclosure) by destroying two endogenous NdeI restriction sites by completing the T4 polymerase end, then blunt-end ligation and replacing the short DNA sequence between the unique ClaI and KpnI restriction sites with the following oligonucleotide with a duplex comprising the sequence SEQ. ID NO .: 1 and SEQ. ID NO .: 2

Clal KpnI 'CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3''TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5 ¹

The plasmid pCFM1156KGF is then cloned into FM5 host cells (ATCC No. 53911) by standard electroporation transformation (using a BioRad Gene Pulser). To reduce the observed internal translation initiation, the KGF DNA sequence between the KpnI and EcoRI restriction sites is replaced with synthetic oligonucleotide DNA that is designed to reduce the use of the internal ribosomal binding site (Fig. 1). After ligation and electroporation into FM5 cells (ATCC No. 53911), a clone containing pCFM1156KGF-dsd was selected. The DNA sequence encoding the KGF gene is confirmed. Thereafter, the cells are cultured at 30 ° C in 10-liter fermenters using standard dosing media to allow cells to grow exponentially. The limiting factor is glucose to prevent the accumulation of toxic by-product. When the semi-exponential cell density is reached, the temperature is raised to 42 ° C for a short time to induce transcription of the KGF gene. Thereafter, the temperature of the fermentation broth is maintained at approximately 42 ° C for the remainder of the KGF induction phase. The separated cell paste is stored frozen. Biologically active KGF is obtained by purification from mechanically lysed cell paste by standard chromatographic procedures using a very high isoelectric point of this protein (S-Sepharose Fast Flow, Pharmacia) and size (Superdex 75, Pharmacia). Purified recombinant KGF protein is subjected to endotoxin testing and mitogenic bioassay described in Rubin et al., PNAS, supra.

The following examples illustrate the invention. The examples are purely illustrative and are not intended to limit the invention in any way.

EXAMPLES OF THE INVENTION

Example 1

KGF-stimulated proliferation and differentiation of adnexal structures in model wound healing in vivo

In this example, a modified partial dermal injury model of the rabbit ear is used.

The rabbit ear ear model dermal described in Mustoe et al., J. Clin. Invest. 87: 694-703 (1991) is modified using a 6mm trephine so that the wound passes through the cartilage to the dermis on the back of the ear. As a result, the wound heals by budding elements of the epithelium under the cartilage. At the same time, the contraction does not become a variable during the healing process, which allows precise quantification of new tissues (see Figure 2).

Recombinant KGF produced as described above is used as a therapeutic agent for wound healing.

Materials and methods

KGF or phosphate buffered saline alone is administered once on the day of surgery and wounds (0.25 cm ² ) are covered with Tegaderm occlusive dressing (manufactured by 3M Company, St. Paul, USA)

MN, USA). Prior to sacrifice, each animal receives an intravenous injection of Brdo (Aldrich Chemical Company, Milwaukee, WI, USA) at 50 mg / kg body weight. Brdu is used to improve the quantification of basal keratinocyte proliferation and basal cell migration kinetics to spinosum and stratum corneum strata. After sacrifice, the injured site is cut into two portions, one portion frozen in Optimum Cooling Temperature Medium (OCT, Miles Inc, Elkhart, IN, USA) and the second portion fixed in Omnifix (Al-Con, Genetics, Inc) Melville, New York, USA) and processes routine histological methods. Staining with Masson Trichrome, Red Oil Red, and IHC staining is done on 3 μιη wound sections.

Reepithelization measurement

For each injury, the total gap and epithelial gap are measured with a calibrated objective micrometer. The percentage of reepithelization in percent of each injury is calculated as the difference between the total gap and the epithelial gap divided by the total gap in the respective injury. The data obtained from the two sections of each injury are averaged. Differences in epithelial gap measurements and reepithelial percentages are analyzed by single-branched single-branch Student t-test.

For each single dose (drug or vehicle) group, the area of the epithelium formed is calculated. One-way ANOVA and Dunnett's t-test are performed for each dose against the control group.

Measurement of epithelial cell proliferation and differentiation

Paraffin-embedded 3 μπ tissue sections are stained with anti-BrdU (Dako Corp. Carpinteria, CA,

USA), avidin-biotin complex (Vector Laboratories Inc., Burlingame, CA, USA) and diaminobenzidine substrate (DAB, Sigma Chemical Co., St. Louis, MO, USA). The sections were leached with 0.1% protease solution and then treated with two normal hydrochloric acid. The endogenous peroxidase is decomposed by treatment with a 3% hydrogen peroxide solution. The preparations are blocked with a 10% normal horse serum solution in phosphate buffered saline (PBS) and then incubated with anti-BrdU diluted 1: 400 with 1% bovine serum albumin (BSA). After washing, the sections are incubated with the peroxidase-bound avidin-biotin complex for 20 minutes at 1 dilution:

100 in 1% BSA solution. Thereafter, the slides are placed in contact with the DAB substrate (10 mg DAB, 20 ml PBS and 20 µl 30% hydrogen peroxide) for 10 minutes. Finally, sections are stained with hernatoxylin.

Histological analysis of BrdU-treated hair follicles

After introduction of BrdU and staining with IHC, the total number of vesicles in the wound bed is determined and the data is analyzed using Kruskall-Wallis multiple comparison test. Further analysis includes a Chi-square test on the percentage of follicle injuries containing 10 or more proliferative cells in each group with one treatment level. In addition, the number of BrdU-positive stained cells is found in all follicles containing more than 5 proliferative cells.

Staining of sebaceous glands with Oil Red o

Oil Red O, which is specific for neutral lipids, is used to identify sebocytes and sebaceous glands. Staining is performed on frozen sections as described by F. L. Carson, Histotechnology: A Self-Instructional Text; ASCP Press, Chicago (1990), USA. Briefly, the frozen sections are air-dried and fixed in zinc formalin for 10 minutes, then the preparations are immersed in a solution of Oil Red o (Sigma Chemical Co., St. Louis, MO, USA) at a concentration of 3 g / liter, further soaked in 60% isopropyl alcohol at room temperature for 30 minutes, decolorized in 60% isopropyl alcohol and stained with Learner hematoxylin. The size of the sebaceous glands and the number of cells in the gland are determined.

Results and discussion

Accelerated reepithelization and increased epithelial thickness are observed in injuries treated with KGF at 4 to 40 µg / cm ² . These lesions show significantly increased reepithelization on day 5 of additional treatment compared to control injuries (76.7% versus 52.5%, 1 μg KGF). The layer of the new epithelium increases in a dose-dependent manner, on day 5 and day 7 of the additional treatment is nearly twice the area of the epithelium in the control injury (at a dose of 10 µg KGF per wound, see Figure 3). Histological analysis shows that thelium epi15 regeneration occurs predominantly by sprouting from adnexal elements in the dermis, ie sebaceous glands, sweat glands and hair follicles.

Analysis of basal keratinocyte proliferation and basal cell migration shows that after the first day of KGF treatment, the number of basal keratinocyte proliferation increases at the margins of the injury (see Figure 4). On the second day of KGF treatment, the number of proliferative keratinocytes in the suprabasal layer of the epidermis is increased, indicating the acceleration of the proliferation of the proliferating cells to the stratum spinosum. On the fifth day of treatment, the wounds show less proliferation in the basal layer, indicating self-limiting acceleration of epithelial repair and differentiation (see Figure 4). Keratinocytes appear to undergo normal maturation during upward migration to form the stratum corneum.

Unexpectedly, increased proliferation of sebocytes and their differentiation, as well as increased proliferation of hair follicles are also observed. Both hair follicles and sebaceous glands appear to be larger and more frequent in KGF injuries compared to control wounds (see Figures 5 and 6). Histological sections stained with Oil Red o for the selective identification of sebaceous glands exhibit a significantly greater number of these glands, and these glands are significantly larger, indicating increased sebocyte proliferation and differentiation to sebum-producing cells. an increase in (depending on the dose) proliferative cells on the hair follicle is also observed.

Recently, epidermal growth factor (EGF) and basic fibroblast growth factor (GFG) (J. Clin. Invest., Quoted above and Pierce et al., Amer. J. Pathol. 140: 1375-1388) have been studied in the rabbit ear model. (1992). Although both growth factors are known to stimulate reepithelization, neither EGF nor basic FGF affect the proliferation or differentiation of adnexal structures such as sebaceous glands and hair follicles. KGF's ability to stimulate proliferation and subsequent differentiation of several epithelial cell types in skin, along with its original fibroblast isolation, indicates that KGF is a potent paracrine stimulator of skin regeneration processes.

Example 2

KGF-stimulated proliferation and differentiation of type II pneumocytes in vivo

This example is performed to evaluate the effect of KGF administration on epithelial cells of the respiratory tract of healthy rats.

Materials and methods

Male Lewis rats weighing 200-250 g are given a single intratracheal injection with varying doses of KGF diluted in saline or phosphate buffered saline using the protocol described in Ulich et al., Amer. J. Pathol. 138: 1485-1496 (1991). KGF is injected at 0.1, 1.0, 5.0 and 10.0 mg / kg. Control rats are administered an intratracheal injection of excipient. At time points 6 hours 1, 2, 3, 4, 5 and 6 days after KGF injection, the rats are sacrificed, their lungs inflated via an intratracheal catheter with Bouin fixative, sagittal sections of the lungs are embedded in paraffin, and histological sections stained with hematoxylin and eoxiline.

Results and discussion

0.1 mg / kg KGF does not induce histologically recognizable alveolar epithelial cell hyperplasia. 1.0 mg / kg KGF elicits a slight but detectable increase in alveolar epithelial cell count. Increased alveolar epithelial cell hyperplasia is determined by dose adjustment to 5.0 and 10.0 mg / kg. KGF does not induce a histologically detectable increase in the number of alveolar epithelial cells at 6 hours and one day after intratracheal injection. At 2 days elevated micropapillary alveolar septal epithelial cells were detected in the lungs of KGF-treated rats (see FIG.

and 8). At 3 days, a diffuse low cube-to-cubic increase in alveolar epithelial cells is found to form a lining of whole alveoli in large lung segments (Figures 9 and 10). However, some lung areas retain normal histology. The histological appearance of the hyperplastic alveolar epithelium in rats on the third day is essentially the same as that of reactive type II pneumocytes in human lungs. The hyperplastic alveolar epithelium is detectable in decreasing amounts in the lungs of KGF-treated rats on days 4 and 5 after intratracheal injection. On day 6, lung rats treated with KGF cannot be distinguished from lungs of control rats.

The lungs of KGF-treated rats were ultrastructively examined 3 days after intratracheal injection. The lining of hyperplastic alveolar epithelial cells in alveolar septa almost always contains one or more lamellar inclusions of varying size and shape (Figures 11 and 12). Bronchial epithelium of KGF-treated rats is hyperplastic on day 3, but this hyperplasia is not immediately as prominent as alveolar cell hyperplasia. Hyperplasia is evaluated as a pseudostratification of epithelial lining of smaller distal bronchi that normally contain one layer of epithelium as a lining, such as bunching or micropapillary growth of the bronchial epithelium and as an increase in mitotic number in the bronchial epithelium (see Figures 13A and 13B).

KGF administered intratracheally induces prominent hyperplasia of alveolar epithelial cells. The presence of lamellar cytoplasmic inclusion in cells at the ultrastructural level is consistent with the assumption that hyperplastic cells are type II pneumocytes. Lamellar inclusions do not appear to be so numerous or so eighth. Inclusions, illustrated in some cases by experimental type II pneumocyte hyperplasia in rats, are very similar to lamellar inclusions illustrated in normal rat lungs. KGF induces not only alveolar cell hyperplasia, but also bronchial epithelial cell hyperplasia. It is possible that the hyperplastic alveolar epithelium represents the undergrowth of the bronchial epithelium from terminal bronchioles to the alveolar parenchyma. However, it seems more likely that KGF acts directly on type II pneumocytes with respect to multifocal micropapillary pneumocyte budding on alveolar septa 2 days after KGF injection, a type of increase that precedes the confluent lining of alveol by cubic cells on day 3. In addition, it is assumed that type II pneumocytes form a mitotic responsive population of alveolar epithelial cells and it is expected that just alveolar cells will be responsive to a potential endogenous alveolar cell growth mediator such as KGF. Finally, the identification of lamellar inclusions in hyperplastic cells confirms not only their differentiation into type II pneumocytes but also the fact that they originate from type II pneumocytes.

The stimulating effect of KGF on type II pneumocytes is consistent with the observation that the amount of KGF receptor messenger RNA in the lung equivalent sample of total RNA is two to three times higher than that found in various other organs of a young adult rat. In this experiment, the KGF receptor messenger RNA level was determined using a modified Ambion RNAse Protection Assay (RPA II,

No. 1410).

The antisense RNA probe used in this assay has the following sequence (SEQ. ID NO .: 3)

5 'GAAUACGAAUUCCUUGCUGUUUGGGCAGGACAGUGAGCCAGGCAGACUGGUUGGCCUGCCCUAUAUAAUUGGAGACCUUACAUA.UAUAUUCCCCAGCAUCCAUCUCCGUCACAUUGAACAGAGCCAGCACUUCUGCAUUGGAGCUAUUUAUCCCCGAGUGGAUC 3'

An antisense probe is made using the Promega Riboprobe Gemini II kit (# P2020) from a linearized template DNA encoding the corresponding DNA sequence described above. The RNA probe is purified on a urea-polyacrylamide gel by excising the correct size band and eluting the RNA probe with Ambion Solution F.

Messenger RNA (standard) encoding a complementary sequence of RNA antisense relative to the probe has the following sequence (SEQ. ID NO .: 4) & GGGAGACAAGCUUGCAUGCCUGCAGGUCGACUCUAGAGGAUCCACUCGGGGAUAAAUAGCÚCCAAUGCAGAAGUGCUGGCUCUGUUCAAUGUGACGGAGAUGGAUGCUGGGGAAUAUAUAUGUAAGGUCUCCAAUUAUAUAGGGCAGGCCAACCAGUCUGCCUGGCUCACUGUCCUGCCCAAACAGCAAGG 3 '

The mediator standard is prepared cold (no radioactive label) using the Promega kit

Riboprobe Gemini II kit (# P2020) and purified on a G50 Sephadex Quick Spin Column.

According to the Ambion standard protocol (RPA II no.

1410), 50 complete RNA is incubated with 10 ⁵ cpm / probe, first 4 minutes at 95 ° C and then 12-18 hours at 45 ° C. RNase (1: 500 Ambion solution R) is incubated with the hybridization solution for 40 minutes at 37 ° C and then an inactivating / precipitating agent (solution Dx) is added.

The RNase-protected fragments are separated by size by urea-polyacrylamide gel electrophoresis and the entire gel is exposed to phosphorimager. The bands are quantified using a Molecular Dynamics Phosphorimager. The results are shown in Figure 14.

Identification of KGF as type II pneumocyte growth factor increases the possibility that KGF will show similar therapeutic potential to glucocorticoids as stimulators of fetal lung maturation. KGF could also be clinically useful in stimulating bronchoalveolar repair after lung injury in adults. The proliferative effect of KGF on type II pneumocytes suggests that KGF could play an important role in the regulation of surfactant synthesis and secretion.

Example 3

KGF-stimulated hepatocyte proliferation and differentiation

In this example, the effects of systemically administered KGF on the epithelial tissues of adult rat liver are evaluated.

Materials and methods

Male rats used as test animals receive water and food ad libitum. All animals receive once daily intraperitoneal injections of KGF or PBS for 1, 4 or 7 days. In all experiments, at least five animals per group are used.

Rats are sacrificed by carbon dioxide asphyxiation and blood is collected for serum chemistry by cardiac puncture immediately after sacrifice. The serum is kept frozen until used in the test. Sera are subjected to standard chemical analysis and analysis for electrolytes. The liver is weighed and its weight is expressed in g / 100 g body weight, i.e. as a percentage of body weight. Tissue samples are placed in 10% buffered formalin for routine histological processing.

Liver sections are stained with hematoxylin and eosin (H and E). One hour before sacrifice, rats are injected intraperitoneally with 50 mg / kg Brdo. Anti-BrdU immunohistochemistry is performed to visualize Brdu incorporation into DNA of replication cells.

Using a calibrated eyepiece grid, the Labeling Index (LI) of the liver is determined. 10 squares per animal are calculated and the average is calculated. Next, the total number of cores and labeled cores per unit area is calculated.

For the standardization of fields believed to represent similar areas of the liver parenchyma, fields adjacent to the portal triad are used for counting.

All data were analyzed using a two-branched Student's double-strand t-test or a one-way ANOVA for multiple group comparisons.

Results and discussion

A dose-response study is performed to identify the effective dose of KGF. KGF is administered by injection every day for 4 days and the organs are removed. Liver weight, expressed as% body weight, differs significantly from control liver weight only at the highest dose of 5 mg / kg per day compared to ANOVA (see Figure 15). Interestingly, compared to control livers, the liver of animals receiving KGF contains serum proteins. cholesterol and triglycerides, which are compounds synthesized in the liver, in substantially greater amounts, both at a KGF of 1 mg / kg and at a KGF dose of 5 mg / kg (see Figure 16).

Animals are analyzed after daily, 4 day or 7 day daily administration of KGF at 5 mg / kg. In a thorough autopsy, it is found that the liver is significantly enlarged at day seven. Liver weight is significantly higher at all time points studied in KGF-treated rats (day 7, KGF 4.08 ± 0.08% versus 3.49 ± 0.06% control, p = 0.0003).

Quantification of Brdu positive nuclei shows a triple-fold increase in KGF-treated rats compared to one-day control (see Figure 7). The mitotic index (number), expressed as the number of mitoses per high energy field, is significantly higher in the one-day treatment compared to the control, which is consistent with the number of Brdo.

These results demonstrate the potent mitogenic and differentiating effects of KGF on the liver. The rapid increase in liver weight after a one-day treatment with KGF together with increased mitotic index and BrdU-positive fraction indicate that KGF has rapid and potent effects on this predominantly epithelial tissue. Although the mitotic effects of KGF disappear rapidly, the weight of the liver, as a percentage of body weight, continues to increase in studies. The increased weight is predominantly due to an increase in the number of cells that were already histologically observable during the one-day treatment and were retained in the four-day and seven-day treatments. Since liver weight gain may increase by more than 2-fold in less than 2 weeks, the discovery of significant differences, starting after one-day treatment, is not surprising.

Elevated albumin levels are not associated with any known disease other than dehydration. KGF is likely to induce elevated serum albumin (and other proteins) due to the increased number of hepatocytes present. Increasing protein synthesis induced by KGF would be of great benefit to patients with end-stage liver disease or cirrhosis of the liver that could save their lives.

Example 4

KGF-stimulated liver cell proliferation and regeneration after partial in vivo hepatectomy

In this experiment, male Spraque-Dawley rats weighing 300-340 g were used as test subjects.

Materials and methods

All rats are subjected to partial hepatectomy (2/3) as described in Higgins and Anderson, Archives of Pathology, Vol. 12 p. 186 (1931). From 8 hours after surgery and then once daily, either PBS (control group) or KGF at 1 mg / kg is injected subcutaneously. Control and KGF-treated rats are weighed and killed 4,

7, 10 or 14 days after surgery. The liver is removed from the animals and weighed.

Results and discussion

It has been found that the treatment of partially hepatectomized KGF rats results in a substantial acceleration of liver mass recovery, both absolute and relative (see Figures 18 and 19). In the KGF group, the liver mass returns to pre-surgery values within 7 days. In contrast, the control group (which was not treated with KGF) did not achieve either absolute or relative liver mass recovery after 14 days.

Example 5

The therapeutic effect of KGF on chemically induced liver toxicity

Male Spraque-Dawley rats weighing 300-340 g are used to evaluate the therapeutic effect of KGF on the liver after chemical toxin introduction.

Administration

Groups of 12 male Spraque-Dawley are given carbon tetrachloride at 1 ml / kg or 2 ml / kg in corn oil on day 0 as vehicle. Each single dose group is divided into two subgroups of 6 rats. Starting 3 hours after administration of carbon tetrachloride and then every day, either subcutaneous injection of PBS or KGF at a dose of 1 mg / kg / day is administered to each subgroup. After 22, 72 and 144 hours after administration of carbon tetrachloride, each rat is sampled and the serum glutamate-oxaloacetate tranaminase (SGOT) is determined.

Results and discussion

As shown in Fig. 20, subcutaneous injection of KGF after oral administration of carbon tetrachloride significantly reduces the serum level of SGOT, a known indicator of liver damage. This effect is particularly evident when administering carbon tetrachloride at a dose of 2 ral / kg on the first day after administration.

Example 6

KGF-stimulated proliferation and differentiation of intestinal mucosal epithelial cells

This example shows the biological effects of KGF on epithelial cells of the intestinal mucosa of healthy rats.

Materials and methods

Male rats used as test animals receive water and food ad libitum. All animals receive once daily intraperitoneal injections of KGF or PBS for 1, 4 or 7 days. In all experiments, at least five animals per group are used.

The rats are sacrificed by carbon dioxide asphyxiation. The main organs are weighed and their weight is expressed in g / 100 g of body weight (percentage of body weight). The gut divides the non-glandular upper gastrointestinal tract, the gastrointestinal tract, the small intestine, and the large intestine. Tissue samples are placed in 10% buffered formalin for routine histological processing.

Sections from all collected organs were stained with hematoxylin and eosin (H and E). One hour before sacrifice, rats are injected intraperitoneally with 50 mg / kg BrdU. By standard methods, anti-BrdU immunohistochemistry is performed to visualize BrdU incorporation into DNA of replication cells. The selected tissues were stained with periodic acid schiff (PAS), alcian blue at pH 2.5 and Masson-Trichrome.

A calibrated eyepiece is used to measure gastric mucosa height and PAS gastric staining depth. Similarly, the length of the villi and the depth of the intestinal crypts in the duodenum and the depth of the colon crypts are measured. To quantify changes in goblet cell production, the number of PAS-positive goblet cells is determined to be 100 μιη starting at the base of the duodenal villi. In all gut regions, measurements are made only on glands or villi that are perpendicular to the underlying muscle. The results are given in μιη, or in the case of small intestinal goblet cells, the average number per unit area. In addition, the number of colon crypts and the number of hyperplastic (double or triple branched) crypts occupying a defined colon length are determined. For each animal, 5 to 10 repeat measurements are taken and the average is calculated.

All data are analyzed using a twofold, two-arm Student's t-test or a multi-group, one-way ANOVA (Statview II, Abacus Concepts, Berkeley, CA, USA).

Results and discussion

First, a dose-response study was performed to identify the effective dose of KGF. KGF is administered by injection every day for 4 days and the organs are removed.

A significant dose response is observed in gastric glands, small intestine and colon (Fig. 15).

Animals are analyzed after daily, 4 day or 7 day daily administration of KGF at 5 mg / kg. In a thorough autopsy, it is found that after a four-day and seven-day treatment, the upper part of the digestive tract is significantly thickened by folds of the mucosa. The upper gastrointestinal tract, gastric glands, duodenum and colon show significant weight gain after four days and seven days of administration, but not after one-day administration. The weight of the gastric glands after a seven-day treatment is 0.65 ± 0.01 g for KGF-treated animals and 0.48 ± 0.02 g for control animals (p = 0.0001).

The non-glandular upper gastrointestinal tract shows significantly increased weight after a four-day and seven-day treatment. Based on histological examination, the scaly, non-glandular upper gastrointestinal epithelium is found to be slightly (after four days of treatment) to be markedly (after seven days of treatment) hyperplastic in animals treated with KGF. The proliferative epithelium suddenly changes from cardia from the normal scaly epithelium of the esophagus to the proliferative epithelium of the upper digestive tract. After a one-day treatment, glandular gastric mucosa is histologically normal. After a four-day and seven-day treatment, histological examination revealed an increased number of goblet cells as open cytoplasm cells. KGF-treated animals for 4 and 7 days show a slight increase in mucosal thickness (Fig. 21). After a seven-day treatment, the mucosal adnexal mucosal layer is expanded and moved to the serosal surface. PAS staining after a four-day and seven-day treatment shows a marked increase in the number and size of PAS positive (mucin-producing goblet cells). With BrdU-stained sections, this layer is further identified as a layer of dividing cells in the gastric glands, showing an increase in proliferative cells during treatment. The thickness of the PAS positive layer of the luminal surface of the gastric mucosa is also significantly increased by the hay after the 4 day and 7 day KGF treatment (Fig. 21).

The small intestine is roughly normal at all time points of the study. PAS staining shows an increased number of mucus producing goblet cells. This difference is significant after four days and seven days of treatment (Fig. 22). The villus height does not differ at any time, but the depth of the crypts is substantially greater after one and seven day treatment (Fig. 22).

After a seven-day treatment, the colon mucosa forms wrinkled folds that significantly increase the total surface area. Hyperplasia indicators, double and triple branched colon crypts, are observed in all groups, including control groups, but most frequent and most noticeable in the short-term treatment groups, i.e., one and four day treatment groups, with the number of hyperplastic rats decreasing with treatment duration (FIG. 23). This corresponds to the depth of the crypts in the colon, which increases with the length of treatment (Fig. 24).

PAS staining and alcian blue shows mucus-producing goblet cells in the colon after KGF treatment. In addition, the number of alcian blue positive cup cells on the luminal surface is higher after seven days of treatment in all animals tested. Animals that had been treated for only 4 days showed a significant increase in PAS positive cells in the upper half and one third of colon crypts.

These results indicate that KGF has potent mitogenic and differentiating effects on the epithelial tissues of the gastrointestinal tract. It is clear from BrdU labeling that KGF initiates cell proliferation in the mucus-forming adnexal layer of the gastric mucosa. These cells form the gastric gland progenitor cells. Upon division, cells from this layer migrate upward towards the lumen of the intestines to form surface cells or down to the serosal surface where they become the cells that form the glandular glands (Toner et al., 1989, Gastrointestinal and Esophageal Pathology, Churchill Livingstone, New York, NY, USA, p. 1328). In addition, KGF promotes selective differentiation of mucosal adnexal cells into cells that move upward and become goblet cells. The total thickness of the gastric mucosa is increased after four days and seven days of treatment. Similar effects are observed in the small and large intestine. KGF promotes increased cell division resulting in elongated crypts through stimulation of antenitor cells. KGF also induces differentiation of dividing crypt cells, as evidenced by increased mucus production and higher number of goblet cells in villi or crypts.

The invention has been described in specific embodiments and examples which are not intended to be limiting thereof. For example, any form of KGF that has substantially the same biological properties as the natural KGF can be used for the disclosed uses. Such forms include purified natural KGF alone, chemical-synthesized KGF and recombinant recombinant KGF using expression systems other than those listed above, as well as analogs, variants, chimeras, etc. based on the natural amino acid sequence. All of these forms can be used in the practice of this invention.

It will also be apparent to those skilled in the art that different host-vector systems can be used to express the KGF-encoding sequence. Such systems include mammalian cell-based systems infected with a virus (e.g., vacciniavir, adenovirus, etc.); virus-based insect cell systems (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or other bacteria other than E. coli that are transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The invention is also not limited to the above systems. The expression elements of these vectors may vary in strength and specificity. Depending on the host-vector system used, any of a number of suitable transcription and translation elements may be used.

After the KGF protein product has been isolated, purified, and after having determined KGF activity using the methods described herein, or other methods known to those skilled in the art, it can be formulated into various pharmaceutical compositions. .

These pharmaceutical compositions typically comprise a suitable, usually chemically defined, carrier or excipient for the therapeutic agent, i.e., KGF as well as other additives, depending on the intended route of administration. The compositions may contain aqueous carriers, or they may be solid phase compositions in which KGF is introduced into non-aqueous carriers such as collagens, hyaluronic acid and various polymers. Such compositions may conveniently be formulated into forms suitable for a variety of routes of administration such as injection, oral, topical, intranasal, and pulmonary administration.

It will also be apparent to those skilled in the art that the amount of KGF dosed will vary depending upon the disease being treated and the route of administration. For example, a dose ranging from 0.01 to 500 mg / kg body weight may be used as a guide for the effective dose. Such a dose may be administered as a single dose or repeatedly depending upon the disease and condition of the patient. The above compositions may be administered in combination with other treatments, such as, for example, the administration of other cytokines and growth factors or other pharmaceutical compositions suitable for the treatment of skin, lung, liver and gastrointestinal disorders.

SEQUENCE LIST (1) GENERAL INFORMATION:

(i) APPLICANT: Amgen Inc.

(ii) TITLE OF THE INVENTION: Therapeutic Uses of Keratinocyte

Growth Factor (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESS: Amgen lne. , (B) STREET: Amgen Center, 1840 DeHavilland Drive (C) LOCATION: Thousand Oaks (D) COUNTRY: California (E) COUNTRY: USA (F) ZIP Code: 91320-1789 (ZIP) (v) MACHINE READY FORM:

(A) MEDIA TYPE: Floppy, 3.5 inch, DS, 2.0Mb (B) COMPUTER: IBM compatible (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: Microsoft Word Version 5.1a (vi) DATA RELATED TO FOR THIS APPLICATION:

(A) APPLICATION NUMBER:

(B) DATE OF ADMINISTRATION: 26.3.1993 (C) CLASSIFICATION:

(2) INFORMATION ABOUT SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHAIN: one chain (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CGATTTGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GGTAC 55 (3) INFORMATION ABOUT SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) CHAIN: one chain (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (4) INFORMATION ABOUT SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 base pairs (B) TYPE: nucleic acid (C) CHAIN: one chain (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GAAUACGAAU UCCUUGCUGU UUGGGCAGGA CAGUGAGCCA GGCAGACUGG

UUGGCCUGCC CUAUAUAAUU GGAGACCUUA CAUAUAUAUU CCCCAGCAUC

CAUCUCCGUC ACAUUGAACA GAGCCAGCAC UUCUGCAUUG GAGCUAUUUA

UCCCCGAGUG GAUC 164 (5) INFORMATION ABOUT SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 191 base pairs (B) TYPE: nucleic acid (C) CHAIN: one chain (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GGGAGACAAG CUUGCAUGCC UGCAGGUCGA CUCUAGAGGA UCCACUCGGG

GAUAAAUAGC UCCAAUGCAG AAGUGCUGGC UCUGUUCAAU GUGACGGAGA

UGGAUGCUGG GGAAUAUAUA UGUAAGGUCU CCAAUUAUAU AGGGCAGGCC

AACCAGUCUG CCUGGCUCAC UGUCCUGCCC AAACAGCAAG G

191 'Goal

Claims (6)

PATENT REQUIREMENTS □ S f'X 9 0 + C η π> j: L L · U 01-1832-95-Ce Ndel KGF-dsd TATGTGCAATGACATGACTCCAGAGCAAATGGCTACAAATGTGAACTGTTCCAGCCCTGA ACACGTTACTGTACTGAGGTCTCGTTTACCGATGTTTACACTTGACAAGGTCGGGACT C C E E E Q Τ V V V V C C C C C C C C C C C C C C C C C C C C C C C C C C C CGCTGTGTGTTCTTCAATACTAATGTACCTTCCTCCCCTATATTCTCACTCTTCTGAGAA RKTRSYDYMEGGDIRVRRLFKpnl | ----------------- synthetic DNA ------------ CTGTCGAACACAGTGGTACCTGCGTATCGACAAACGCGGCAAAGTCAAGGGCACCCAAGA GACAGCTTGTGTCACCATGGACGCATAGCTGTTTGCGCCGTTTCAGTTCCCGTGGGTTCT C R T Q W Y L R IDKRGKVKGTQE-: GATGAAAAACAACTACAATATTATGGAAATCCGTACTGTTGCTGTTGGTATCGTTGCAAT CTACTTTTTGTTGATGTTATAATACCTTTAGGCATGACAACGACAACCATAGCAACGTTA MKNNYNIMEIRTVAVGIVAIEcoRI CAAAGGTGTTGAATCTGAATTCTATCTTGCAATGAACAAGGAAGGAAAACTCTATGCAAA GTTTCCACAACTTAGACTTAAGATAGAACGTTACTTGTTCCTTCCTTTTGAGATACGTTT K C- V Ξ S Ξ FYLA mn _kegk LYAK GAAAGAATGCAATGAAGATTG7AACTTCAAAGAACTAATTCTGGAAAACCAT7ACAACAC CTTTCTTACGTTACTTCTAACATTGAAGTTTCTTGATTAAGACCTTTTGGTAATGTTGTG KRCNEDCNFKELILENKYNTATATGCATCAGCTAAATGGACACACAACGGAGGGGAAATGTTTGTTGCCTTAAATCAAAA TATACGTAGTCGATTTACCTGTGTGTTGCCTCCCCTTTACAAACAACGGAATTTAGTTTT YASAKWTKNGGEMFVALNQKGGGGATTCCTGTAAGAGGAAAAAAAACGAAGAAAGAACAAAAAACAGCCCACTTTCTTCC CCCCTAAGGACATTCTCCTTTTTTTTGCTTCTTTCTTGTTTTTTGTCGGGTGAAAGAAGG G I PVRGKKTKKEQKTAKFLPTATGGCAATAACTTAATAG ATACCGTTATTGAATTATCCTAG N A 1 T BamHI FIG. 1 WHAT CTÍ iJ FIG. 2 Epithelial Area (mm ² ) 10.0 pg 0.0044 '•' ll ' Br Fig. —Ίο A — cn (3 OJ ‘ Basal Cell Proliferation / mm i: ‘‘i3 7 '' · 7], '· · i; J \ t GVy 0 o σ hi 120 30 48 120 Post-injury time (h) Suprabasal cell proliferation / mm S S | X 9 o Effect of rKGF on Keratinocytes A method of in vitro stimulating the production of non-keratinocyte epithelial cells, characterized by:. , 16! u L ¹ * that these cells are contacted with an effective amount of keratinocyte growth factor. i 'f'2 2. The method of claim 1, wherein the non-keratinocyte epithelial cells are selected from the group consisting of sebocytes, hair follicle cells, hepatocytes, type II pneumocytes, mucin-producing goblet cells and other epithelial cells and their progenitors in the skin, lungs , liver and gastrointestinal tract. 3 0C i ί 6 f _LL I3B -'iOiMlSV- ^ 3A0? $ Ággw θ ^ Ο FIG. 6 liX 9 o oisoa ? f L i <i f · □ KGFRmRNA equivalent (pg) to 5yg full RNA FIG. 14 depicting ° H3AO'3 ^ 3yd ovy q ^s 5 l! X go 0Ί £ OG ¹ ? ι í. L Ί,% body weight 3. The method of claim 1 wherein the keratinocyte growth factor is recombinant keratinocyte growth factor. 4. The method of claim 3 wherein the recombinant keratinocyte growth factor produced in bacterial cells is used. 5 Γ / 9 0 οΐ5οα ΐ 6 ϊ L L '• f * o FIG. 5 FIG I> ct 6 IIX 9 o 'οί. FIG S δ Π 9 0 0) 330 i?, Í l i l Γ'Ο 6 | χ 9, ' 5. The method of claim 4, wherein E. coli cells are used as bacterial cells. 6. Keratinocyte growth factor for the treatment of dermal injuries in order to stimulate the production of sebaceous gland cells and hair follicles. 7. Keratinocyte growth factor for the treatment of burns. 8. Keratinocyte growth factor for the treatment of epidermolysis bullosa. 9. Keratinocyte growth factor for the treatment of chemotherapy-induced alopecia. 10. Growth male type. factor keratinocytes for treatment plesatos 11. Growth them ulcers. factor keratinocytes for treatment stomach- 12. Growth of ulcer ulcers. factor keratinocytes for treatment twelve- 13. Growth stomach and esophagus. factor keratinocytes for treatment erosion 14. Growth factor keratinocytes for treatment erosive gastritis, oesophagitis or oesophageal reflux. diseases Keratinocyte growth factor for intestine. inflammation16. Keratinocyte growth factor for the treatment or prevention of intestinal toxicity induced by radiation or radiation and chemotherapy. 17. Keratinocyte growth factor for treating hyaline membrane disease. 18. respiratory Keratinocyte growth factor for the treatment of epithelial necrosis induced by smoke inhalation. 19. Keratinocyte growth factor for the treatment of emphysema 20. Keratinocyte growth factor for the treatment of pulmonary inflammation and fibrosis. 21. Keratinocyte growth factor for the treatment of liver. Keratinocyte Growth Factor for the Treatment of Nervous Liver Failure. 23. Keratinocyte growth factor for the treatment of viral hepatitis. cirrhosy fulmiakutní 6 liX AND I CHSOC I: T'2% body weight ΠΛ1 3 1M. CH 3 / .0 ^_ S, total protein Globulin Albumin 'Cholesterol Triglycerides -A ί ί Λί ± S 77Λ OH 3 Λ 0 'i Ai / J Q avy n With 5% o and a% of Brd labeled hepatocytes cr H! after ω ja io: .usvl · * ΰη ^ ΛΡ 3 λίΛ ^{* 1} Q'dc avy O With 5 | 'X' 9 c ci§o I 6 I L L i T'3 FIG. 17B Mitotic number per HPF AND FIG. 18 Liver weight (b) o n < H · CD * O i £) \ t X * iQ l-i ro iQ (D (D i-i n H · (+ ro κ i__ ’Vdd JA i i'Hsv-j * CH3AP '; SÁW QHd gvb o i - *. with 5 j'x 9 o I 0150a, m i i « Liver weight (g) / 100 g body weight Day O n < H3 ro * o * 3 l · - * in £ 3 a ro tu; -i ro ro κ o n Htu rt ro hf iidd ^J '^ INLSVTA OhgAC!' * o cr Hi ^{with 6} l! X 9 o OI5OQ ΐ? Γ i) 0 <0 SGOT by JX 9C? Π, Π » I? ' L L L ‘1 i 'f3 Days after administration of CCI FIG. 20 FIG. 21A FIG. 21B The length of the gastric glands (^ um) in IΛ with 6 | X 9 o 01500 ΐ Z I L L 'f'2 O O O O o o o Color Depth of PAS (yUm) Depth of duodenal crypts (yum) o cr pi to it> o σ i-i it to ω ro IOINiSV1A avy n S δ | 'X' 9 o I 7- í ^: LL ¹¹ • f'3 o cn o Number of cells in vials stained with PAS / 100 / µm L'i i i V V Ί Η ΛΓ 3 A y y 4 4 4 vy With S | iX '9 0 C'5OG FIG. 23 ί '; i -> j A i O! N i S V1Λ OA 3 AA o.sAydd _(σ / or O s i · S 5 liX 9 0 .. . I i Qi§06! , í 6 LL LL ^{! 1} J FIG. 24